Illuminating Airway Nerve Structure and Function in Chronic Cough.

Airway nerves regulate vital airway functions including bronchoconstriction, cough, and control of respiration. Dysregulation of airway nerves underlies the development and manifestations of airway diseases such as chronic cough, where sensitization of neural pathways leads to excessive cough triggering. Nerves are heterogeneous in both expression and function. Recent advances in confocal imaging and in targeted genetic manipulation of airway nerves have expanded our ability to visualize neural organization, study neuro-immune interactions, and selectively modulate nerve activation. As a result, we have an unprecedented ability to quantitatively assess neural remodeling and its role in the development of airway disease. This review highlights our existing understanding of neural heterogeneity and how advances in methodology have illuminated airway nerve morphology and function in health and disease.

Grandmaternal allergen sensitization reprograms epigenetic and airway responses to allergen in second-generation offspring.

Asthma susceptibility is influenced by environmental, genetic, and epigenetic factors. DNA methylation is one form of epigenetic modification that regulates gene expression and is both inherited and modified by environmental exposures throughout life. Prenatal development is a particularly vulnerable time period during which exposure to maternal asthma increases asthma risk in offspring. How maternal asthma affects DNA methylation in offspring and what the consequences of differential methylation are in subsequent generations are not fully known. In this study, we tested the effects of grandmaternal house dust mite (HDM) allergen sensitization during pregnancy on airway physiology and inflammation in HDM-sensitized and challenged second-generation mice. We also tested the effects of grandmaternal HDM sensitization on tissue-specific DNA methylation in allergen-naïve and -sensitized second-generation mice. Descendants of both allergen- and vehicle-exposed grandmaternal founders exhibited airway hyperreactivity after HDM sensitization. However, grandmaternal allergen sensitization significantly potentiated airway hyperreactivity and altered the epigenomic trajectory in second-generation offspring after HDM sensitization compared with HDM-sensitized offspring from vehicle-exposed founders. As a result, biological processes and signaling pathways associated with epigenetic modifications were distinct between lineages. A targeted analysis of pathway-associated gene expression found that Smad3 was significantly dysregulated as a result of grandmaternal allergen sensitization. These data show that grandmaternal allergen exposure during pregnancy establishes a unique epigenetic trajectory that reprograms allergen responses in second-generation offspring and may contribute to asthma risk.NEW & NOTEWORTHY Asthma susceptibility is influenced by environmental, genetic, and epigenetic factors. This study shows that maternal allergen exposure during pregnancy promotes unique epigenetic trajectories in second-generation offspring at baseline and in response to allergen sensitization, which is associated with the potentiation of airway hyperreactivity. These effects are one mechanism by which maternal asthma may influence the inheritance of asthma risk.

The amniotic fluid proteome changes across gestation in humans and rhesus macaques.

Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC-MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies.

From bench to bedside: The role of cough hypersensitivity in chronic cough.

BACKGROUND: Chronic cough is a burdensome condition characterized by persistent cough lasting longer than 8 weeks. Chronic cough can significantly affect quality of life, physical function and productivity, with many people troubled with a cough that lasts for months or even years. People with chronic cough commonly report a persistent urge to cough with frequent bouts of coughing triggered by innocuous stimuli, which has led to the concept of cough hypersensitivity. MAIN BODY: Both central and peripheral neural pathways regulate cough, and although mechanisms driving development of cough hypersensitivity are not fully known, sensitization of these neural pathways contributes to excessive cough triggering in cough hypersensitivity. Effective therapies that control chronic cough are currently lacking. Recent therapeutic development has focused on several ion channels and receptors involved in peripheral activation of cough (e.g., transient receptor potential channels, P2 × 3 receptors and voltage-gated sodium channels) or central cough processing (e.g., neurokinin-1 [NK-1] receptors and nicotinic acetylcholine receptors). CONCLUSION: These targeted therapies provide novel insights into mechanisms underlying cough hypersensitivity and may offer new treatment options for people with chronic cough. In this review, we explore preclinical and clinical studies that have improved our understanding of the mechanisms responsible for chronic cough and discuss the most promising targeted approaches to date, including trials of P2 × 3-receptor antagonists and NK-1-receptor antagonists.

Unique Allergic Asthma Phenotypes in Offspring of House Dust Mite-exposed Mice.

Asthma is a heterogeneous inflammatory airway disease that develops in response to a combination of genetic predisposition and environmental exposures. Patients with asthma are grouped into phenotypes with shared clinical features and biomarker profiles to help tailor specific therapies. However, factors driving development of specific phenotypes are poorly understood. Prenatal exposure to maternal asthma is a unique risk factor for childhood asthma. Here we tested whether maternal asthma skews asthma phenotypes in offspring. We compared airway hyperreactivity and inflammatory and neurotrophin lung signatures before and after allergen challenge in offspring born to mice exposed to house dust mite (HDM) or vehicle during pregnancy. Maternal HDM exposure potentiated offspring responses to HDM allergen, significantly increasing both airway hyperreactivity and airway eosinophilia compared with control mice. Maternal HDM exposure broadly skewed the offspring cytokine response from a classic allergen-induced T-helper cell type 2 (Th2)-predominant signature in HDM-treated offspring of vehicle-exposed mothers, toward a mixed Th17/Th1 phenotype in HDM-treated offspring of HDM-exposed mothers. Morphologic analysis determined that maternal HDM exposure also increased airway epithelial sensory nerve density and induced distinct neurotrophin signatures to support airway hyperinnervation. Our results demonstrate that maternal allergen exposure alters fetal lung development and promotes a unique inflammatory phenotype at baseline and in response to allergen that persists into adulthood.

Multicolor labeling of airway neurons and analysis of parasympathetic heterogeneity.

We report subpopulations of airway parasympathetic neurons expressing substance P, neuronal nitric oxide synthase, and tyrosine hydroxylase, highlighting unexplored heterogeneity in this population. These neurotransmitter-specific subpopulations did not form intraganglionic interneurons, but rather, extended outside the ganglia, into the airways, to distant innervation targets. Our experiments demonstrate the utility of multicolor labeling to characterize airway innervation, allowing us to confirm the extensive heterogeneity of postganglionic parasympathetic neurons. These methods will facilitate future investigations of neurophysiology and neural contributions to airway disease.

Eosinophils in Health and Disease: A State-of-the-Art Review.

Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

TLR7 is expressed by support cells, but not sensory neurons, in ganglia.

BACKGROUND: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. METHODS: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. RESULTS: In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. CONCLUSION: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7's neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7's primary role in neuronal tissues is not related to antiviral immunity.

Airway Sensory Nerve Plasticity in Asthma and Chronic Cough.

Airway sensory nerves detect a wide variety of chemical and mechanical stimuli, and relay signals to circuits within the brainstem that regulate breathing, cough, and bronchoconstriction. Recent advances in histological methods, single cell PCR analysis and transgenic mouse models have illuminated a remarkable degree of sensory nerve heterogeneity and have enabled an unprecedented ability to test the functional role of specific neuronal populations in healthy and diseased lungs. This review focuses on how neuronal plasticity contributes to development of two of the most common airway diseases, asthma and chronic cough, and discusses the therapeutic implications of emerging treatments that target airway sensory nerves.

Onset of Skin, Gut, and Genitourinary Prodromal Parkinson's Disease: A Study of 1.5 Million Veterans.

BACKGROUND: Prodromal Parkinson's disease of skin, genitourinary, and gastrointestinal systems offers a unique window for understanding early disease pathogenesis and developing disease modifying treatments. However, prior studies are limited by incomplete timing information, small sample size, and lack of adjustment for known confounders. Verifying prodromal timing and identifying new disorders in these accessible organs is critically important given their broad use. OBJECTIVE: We aimed to measure onset timing for gastrointestinal, genitourinary, and skin disorders in a large, nationwide clinically characterized cohort of 1.5 million participants. METHODS: Patients with Parkinson's disease (n = 303,693) were identified using diagnostic codes in the medical records database of the United States Veterans Affairs healthcare system and were compared 4:1 with matched controls. Disorder prevalence and estimated onset times were assessed for 20 years preceding diagnosis. RESULTS: The earliest significantly increased prodromal disorders were gastroesophageal reflux, sexual dysfunction, and esophageal dyskinesia at 17, 16, and 15 years before diagnosis. Estimated onset times for each disorder occurred 5.5 ± 3.4 years before the first measured increase. The earliest estimated onset times were smell/taste, upper gastrointestinal tract, and sexual dysfunction at 20.9, 20.6, and 20.1 years before diagnosis. Onset times for constipation and urinary dysfunction were notably longer by 7 and 9 years compared to prior studies in sleep disorder patients. Dermatophytosis and prostatic hypertrophy were identified as new high prevalence prodromal disorders. CONCLUSIONS: Gastrointestinal, genitourinary, and skin disorders manifest decades before diagnosis of Parkinson's disease, reiterating their potential as sites for developing early diagnostic testing and understanding pathogenesis.

Interactions of Eosinophils with Nerves.

Eosinophils affect nerve structure and function in organs such as lungs and skin, which contributes to disease pathogenesis. We have developed methods for culturing primary sensory and parasympathetic neurons in multiple species and have refined these techniques for coculture with eosinophils. Eosinophil-nerve coculture has been an essential tool for testing interactions between these cell types. Here we describe methods for coculturing primary parasympathetic ganglia, vagal sensory nerves, and dorsal root sensory nerves with eosinophils.

Airway Sensory Nerve Density Is Increased in Chronic Cough.

Rationale: Chronic cough is characterized by frequent urges to cough and a heightened sensitivity to inhaled irritants. Airway sensory nerves trigger cough. We hypothesized that sensory nerve density is increased in chronic cough, which may contribute to excessive and persistent coughing.Objectives: To measure airway nerve density (axonal length) and complexity (nerve branching, neuropeptide expression) in humans with and without chronic cough.Methods: Bronchoscopic human airway biopsies were immunolabeled for nerves and the sensory neuropeptide substance P. Eosinophil peroxidase was also quantified given previous reports showing associations between eosinophils and nerve density. Three-dimensional image z-stacks of epithelium and subepithelium were generated using confocal microscopy, and from these z-stacks, total nerve length, the number of nerve branch points, substance P expression, and eosinophil peroxidase were quantified within each airway compartment.Measurements and Main Results: Nerve length and the number of branch points were significantly increased in epithelium, but not subepithelium, in chronic cough compared with healthy airways. Substance P expression was scarce and was similar in chronic cough and healthy airways. Nerve length and branching were not associated with eosinophil peroxidase nor with demographics such as age and sex in either group.Conclusions: Airway epithelial sensory nerve density is increased in chronic cough, suggesting sensory neuroplasticity contributes to cough hypersensitivity.

Transient receptor potential ankyrin-1 causes rapid bronchodilation via nonepithelial PGE2.

Transient receptor potential ankyrin-1 (TRPA1) is a ligand-gated cation channel that responds to endogenous and exogenous irritants. TRPA1 is expressed on multiple cell types throughout the lungs, but previous studies have primarily focused on TRPA1 stimulation of airway sensory nerves. We sought to understand the integrated physiological airway response to TRPA1 stimulation. The TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) were tested in sedated, mechanically ventilated guinea pigs in vivo. Reproducible bronchoconstrictions were induced by electrical stimulation of the vagus nerves. Animals were then treated with intravenous AITC or CINN. AITC and CINN were also tested on isolated guinea pig and mouse tracheas and postmortem human trachealis muscle strips in an organ bath. Tissues were contracted with methacholine, histamine, or potassium chloride and then treated with AITC or CINN. Some airways were pretreated with TRPA1 antagonists, the cyclooxygenase inhibitor indomethacin, the EP2 receptor antagonist PF 04418948, or tetrodotoxin. AITC and CINN blocked vagally mediated bronchoconstriction in guinea pigs. Pretreatment with indomethacin completely abolished the airway response to TRPA1 agonists. Similarly, AITC and CINN dose-dependently relaxed precontracted guinea pig, mouse, and human airways in the organ bath. AITC- and CINN-induced airway relaxation required TRPA1, prostaglandins, and PGE2 receptor activation. TRPA1-induced airway relaxation did not require epithelium or tetrodotoxin-sensitive nerves. Finally, AITC blocked airway hyperreactivity in two animal models of allergic asthma. These data demonstrate that stimulation of TRPA1 causes bronchodilation of intact airways and suggest that the TRPA1 pathway is a potential pharmacological target for bronchodilation.

Inflammatory mechanisms linking maternal and childhood asthma.

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness, inflammation, and remodeling. Asthma often develops during childhood and causes lifelong decrements in lung function and quality of life. Risk factors for childhood asthma are numerous and include genetic, epigenetic, developmental, and environmental factors. Uncontrolled maternal asthma during pregnancy exposes the developing fetus to inflammatory insults, which further increase the risk of childhood asthma independent of genetic predisposition. This review focuses on the role of maternal asthma in the development of asthma in offspring. We will present maternal asthma as a targetable and modifiable risk factor for childhood asthma and discuss the mechanisms by which maternal inflammation increases childhood asthma risk. Topics include how exposure to maternal asthma in utero shapes structural lung development with a special emphasis on airway nerves, how maternal type-2 cytokines such as IL-5 activate the fetal immune system, and how changes in lung and immune cell development inform responses to aero-allergens later in life. Finally, we highlight emerging evidence that maternal asthma establishes a unique "asthma signature" in the airways of children, leading to novel mechanisms of airway hyperreactivity and inflammatory cell responses.

Development of a National Academic Boot Camp to Improve Fellowship Readiness.

Background: Pulmonary and critical care medicine (PCCM) fellowship requires a high degree of medical knowledge and procedural competency. Gaps in fellowship readiness can result in significant trainee anxiety related to starting fellowship training.Objective: To improve fellowship readiness and alleviate anxiety for PCCM-bound trainees by improving confidence in procedural skills and cognitive domains.Methods: Medical educators within the American Thoracic Society developed a national resident boot camp (RBC) to provide an immersive, experiential training program for physicians entering PCCM fellowships. The RBC curriculum is a 2-day course designed to build procedural skills, medical knowledge, and clinical confidence through high-fidelity simulation and active learning methodology. Separate programs for adult and pediatric providers run concurrently to provide unique training objectives targeted to their learners' needs. Trainee assessments include multiple-choice pre- and post-RBC knowledge tests and confidence assessments, which are scored on a four-point Likert scale, for specific PCCM-related procedural and cognitive skills. Learners also evaluate course material and educator effectiveness, which guide modifications of future RBC programs and provide feedback for individual educators, respectively.Results: The American Thoracic Society RBC was implemented in 2014 and has grown annually to include 132 trainees and more than 100 faculty members. Mean knowledge test scores for participants in the 2019 RBC adult program increased from 55% (±14% SD) on the pretest to 72% (±11% SD; P < 0.001) after RBC completion. Similarly, mean pretest scores for pediatric course attendees increased from 54% (±13% SD) to 62% (±19% SD; P = 0.17). Specific content domains that improved by 10% or more between pre- and posttests included airway management, bronchoscopy, pulmonary function testing, and code management for adult course participants, and airway management, pulmonary function testing, and extracorporeal membrane oxygenation for pediatric course participants. Trainee confidence also significantly improved across all procedural and cognitive domains for adult trainees and in 10 of 11 domains for pediatric course attendees. Course content for the 2019 RBC was overwhelmingly rated as "on target" for the level of learner, with <4% of respondents indicating any specific session was "much too basic" or "much too advanced."Conclusion: RBC participation improved PCCM-bound trainee knowledge, procedural familiarity, and confidence. Refinement of the RBC curriculum over the past 7 years has been guided by educator and course evaluations, with the ongoing goal of meeting the evolving educational needs of rising PCCM trainees.

IL-5 Exposure In Utero Increases Lung Nerve Density and Airway Reactivity in Adult Offspring.

Asthma is characterized by airway hyperreactivity and inflammation. In the lungs, parasympathetic and sensory nerves control airway tone and induce bronchoconstriction. Dysregulation of these nerves results in airway hyperreactivity. Humans with eosinophilic asthma have significantly increased sensory nerve density in airway epithelium, suggesting that type 2 cytokines and inflammatory cells promote nerve growth. Similarly, mice with congenital airway eosinophilia also have airway hyperreactivity and increased airway sensory nerve density. Here, we tested whether this occurs during development. We show that transgenic mice that overexpress IL-5, a cytokine required for eosinophil hematopoiesis, give birth to wild-type offspring that have significantly increased airway epithelial nerve density and airway hyperreactivity that persists into adulthood. These effects are caused by in utero exposure to maternal IL-5 and resulting fetal eosinophilia. Allergen exposure of these adult wild-type offspring results in severe airway hyperreactivity, leading to fatal reflex bronchoconstriction. Our results demonstrate that fetal exposure to IL-5 is a developmental origin of airway hyperreactivity, mediated by hyperinnervation of airway epithelium.